Negative selection and regulatory T cell development are required to protect against organ-specific autoimmunity, to prevent inappropriate responses to commensal microorganisms, and to help terminate immune responses to infections following clearance of the pathogen. Recent studies have shown that regulatory T cells develop from two distinct progenitor subsets - a CD25+FOXP3- Treg progenitor and a CD25- FOXP3lo Treg progenitor. A fundamental question is why there should be two distinct pathways to reach ostensibly the same output (mature Tregs). Our central hypothesis is that these two Treg progenitor populations yield functionally distinct Treg subsets with specialized roles in tolerance A second key question is how the Treg lineage is selected such that its TCR repertoire is skewed in favor of higher self-reactivity. Our preliminary data document that expression of three TNF receptor superfamily (TNFRSF) members - GITR, OX40 and TNFR2 - correlates directly with TCR signal strength and is required for Treg development. Further, our data suggest that GITR, OX40 and TNFR2 also influence TCR-dependent negative selection. In our model TNFRSF signaling primes CD4+ thymocytes for cell death as well as responsiveness to IL2. Those cells that subsequently receive an IL2 signal differentiate into Tregs while those that fail to bind IL2 undergo clonal deletion. Based on these data, we postulate that TCR signal strength is directly linked to expression of GITR, OX40 and TNFR2 and that signaling via these TNFRSF members primes developing thymocytes for both negative selection and Treg development. The result of this molecular programming is preferential development of a Treg TCR repertoire biased towards higher self-reactivity. Thus, the studies proposed in this grant application will elucidate how specific TNFRSF members link TCR signals to the establishment of a functional Treg pool and help broaden the repertoire of thymocytes undergoing negative selection.